1. Field of the Invention
This invention relates to the field of molecular biology and, in particular, to nucleic acid molecules encoding an Rb-interacting zinc finger (RIZ) protein and a conserved domain of a RIZ protein that is involved in regulating gene transcription.
2. Background Information
The retinoblastoma Rb protein is known to play a key role in controlling normal cell proliferation and differentiation. The ability of a cell to divide requires the cell to pass through the various phases of the cell cycle. Although Rb is believed to keep normal cells from dividing by maintaining them in a phase of the cell cycle known as G1 or G0, the precise mechanism underlying Rb function is unknown. It is known, however, that Rb can bind various cellular proteins, including proteins involved in regulating gene transcription. Thus, Rb may exert its action by interacting with such cellular proteins.
The role that Rb plays in controlling cell growth makes it an attractive target for promoting the growth of tissues that normally do not grow because of the action of Rb. For example, cardiac muscle tissue and nervous tissue that have lost function due to cell death are not usually repaired by subsequent proliferation of the remaining live cells. Thus, a method to block the growth controlling function of Rb can be useful for inducing tissue repair in situations of cardiac or neural cell death.
Rb also is known as a tumor suppressor since the abnormal growth of a cancer cell can result from inactivation of Rb protein. Such inactivation can occur either due to a mutation or to inactivation of Rb protein subsequent to binding a viral oncoprotein, a product of an oncogenic tumor virus. A particular region in Rb called the Rb pocket appears to be critical for its growth controlling function since Rb inactivation by mutation or by oncoprotein binding impacts this region.
The importance of the Rb pocket in the functioning of Rb and the understanding that viral oncoproteins can regulate Rb by binding the pocket suggest that there may be normal cellular proteins that can regulate the function of Rb by binding the pocket. The identification of such proteins will provide new approaches to regulate the control of cell proliferation mediated by Rb in diseases such as those that involve loss of cardiac or neural function or in the control of cancer.
Thus, a need exists to identify proteins that can bind to and regulate Rb in order to provide new approaches for controlling cell proliferation and differentiation. The present invention satisfies this need and provides related advantages as well.
The present invention provides substantially purified mammalian Rb-interacting zinc finger proteins (RIZ), including for example, human RIZ and rat RIZ. In addition, the invention provides active fragments of a RIZ such as the sequences EIRCEEKPEDL (SEQ ID NO: 6) and EIRCDEKPEDL (SEQ ID NO: 91), which bind Rb. The invention also provides antibodies that can specifically bind to a RIZ or a mutant RIZ.
The invention further provides nucleic acid molecules encoding mammalian RIZ and active fragments thereof, vectors containing the nucleic acid molecules and host cells containing the vectors. In addition, the invention provides nucleotide sequences that can specifically hybridize to a nucleic acid molecule encoding a RIZ or a mutant nucleic acid molecule encoding a RIZ.
In addition, the invention provides a peptide comprising a PR domain, which is conserved among various proteins and can be involved in regulating the transcription of a target gene. In general, a PR domain peptide contains about 100 to about 120 amino acids that characteristically are arranged as a series of three highly conserved sequences of about ten to about twelve amino acids each, which are separated from each other by less conserved sequences of about 24 to about 34 amino acids each. A PR domain of the invention is exemplified by the PR domain present in the RIZ protein disclosed herein and by the PR domain present in the proteins PRDI-BF1, Evi-1 and egl-43.
The present invention further provides fusion proteins comprising a PR domain of the invention operably linked to a peptide that can bind to a DNA regulatory element. For example, a fusion protein of the invention can comprise a PR domain operably linked to a peptide that binds to a particular gene promotor or enhancer, wherein binding of the fusion protein to a target gene, which is a gene containing the particular promotor or enhancer, can alter expression of the target gene. Thus, a fusion protein of the invention can be useful for regulating the transcription of one or more target genes.
In addition, the invention further provides methods of identifying transcription factors and oncogenic. proteins that bind a PR domain peptide or a RIZ active fragment containing a PR domain. The identification of such factors and proteins provides new approaches to manipulate cell differentiation and transformation.
The invention also provides a screening assay useful for identifying agents that can effectively alter the association of a RIZ with a second molecule such as Rb or can effectively alter the activity of a RIZ. By altering the association of a RIZ with a second molecule or altering the activity of a RIZ, an effective agent can modulate a function of a cell such. as cell proliferation.
The invention further provides methods for promoting the growth of a cell such as a neural cell or cardiac muscle cell by contacting the cell with an effective agent. For example, cell growth can be promoted by introducing into a cell an effective agent such as an expression vector having an expression control sequence operably linked to a nucleotide sequence encoding an active fragment of a RIZ, wherein the active fragment lacks the growth-suppressing properties of a complete RIZ protein. In addition, the invention provides methods for restoring normal controlled cell growth to cancer cells by introducing into the cancer cells an expressible nucleic acid molecule encoding a complete RIZ protein.
The invention also provides methods of detecting a RIZ in a sample by detecting the presence of the RIZ protein or of a nucleic acid molecule encoding the RIZ. Such methods can be used to diagnose a pathology characterized by an increased or decreased level of expression of a RIZ in a cell or by expression of a mutant RIZ. Such a method also can be used to diagnose a pathology characterized by a mutant nucleic acid molecule encoding a RIZ.
The invention further provides methods useful for isolating Rb tumor suppressor protein or a mutant Rb from a sample. For example, Rb can be isolated from a sample by affinity chromatography using a RIZ or a RIZ active fragment such as the sequences EIRCEEKPEDL (SEQ ID NO: 6) or EIRCDEKPEDL (SEQ ID NO: 91).